摘要
目的:研究TGF-β1在黑色素瘤细胞中的作用,为可能的黑色素瘤分子靶向治疗提供理论依据。方法:分别收集黑色素瘤病人与正常人血清,去除Ig G、白蛋白等高丰度蛋白,液相分离后质谱检测,筛选血清差异蛋白。筛选出TGF-β1后进行ELISA验证。进一步构建TGF-β1稳定高表达的黑色素瘤细胞(A-375及SK-MEL-1),利用荧光定量PCR和Western blot进行验证。采用PIAnnexin V双染及流式细胞术分析TGF-β1高表达对黑色素瘤细胞凋亡的影响。结果:与正常人血清对照组比较,共鉴定了84个黑色素瘤相关的血清差异蛋白,其中33个蛋白上调,49个蛋白下调(TGF-β1明显下调),ELISA结果验证了TGF-β1在黑色素瘤病人血清中表达水平相对于健康对照下调2.3倍(P<0.05)。荧光定量PCR和Western blot验证显示构建的TGF-β1稳定高表达的黑色素瘤细胞A-375及SK-MEL-1中TGF-β1 m RNA和蛋白均较对照A-375及SK-MEL-1细胞明显升高(P<0.05或P<0.01);与正常对照组细胞比较,TGF-β1高表达时A-375及SK-MEL-1细胞凋亡率均升高(P均<0.01)。结论:TGF-β1在黑色素瘤病人中表达水平显著降低,当其表达水平升高时可诱导黑色素瘤细胞凋亡,提示TGF-β1可能作为黑色素瘤治疗的潜在靶分子。
OBJECTIVE： Melanoma usually has poor prognosis ,thus the aims of this study were to find potential targets of melanoma in serum and to understand their roles in the progression of melanoma. METHODS ：Serum samples from patients and healthy controls were collected. From the samples＇IgG and albumin were removed＇and proteins were digested and labeled with iTRAQ labeling reagent. Differentially expressed serum proteins were identified by LC-MS/MS and validated by ELISA. TGF-β1 overexpressed melanoma cells were constructed using PLVX plasmid and lentivirus transfection. Cell apoptosis were analyzed by flow cytomeric assay. RESULTS ：81 melanoma-associated proteins were identified＇33 were up-regulated and 49 were down-regulated. TGF-β1 was found significantly down-regulated （3.17± 0.76, P〈0.05） in melanoma patients’ serum and validated by ELISA. TGF-β1 overexpressed melanoma cells （A-375 and SK-MEL-1） were constructed＇the apoptosis analysis showed that TGF-β1 could significantly induced apoptosis of melanoma cells. CONCLUSION ：TGF-β1 was reduced in melanoma patients’ serum and was involved with inhibition of tumor cell proliferation. Thus＇the findings suggest that TGF -β1 could be an important potential target in melanoma.
出处
《癌变．畸变．突变》
CAS
CSCD
2016年第5期348-352,共5页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
深圳市知识创新计划基础研究项目(JCY20140411092959833)
深圳市科技计划项目(ZDSYS20140509150415945)
深圳市科技创新委基础研究学科布局项目(JCYJ20160328161613864)
